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The Researcher's Guide to Measuring Binding Affinity and Why It Matters

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The Researcher's Guide to Measuring Binding Affinity and Why It Matters The Researcher’s Guide to Measuring Binding Affinity and Why It Matters About this eBook Find out the key principles for measuring binding affinity, including the critical parameters to consider and what solutions are available. Whether you’re just starting to learn about binding interactions or you want a refresher, this guide will provide helpful tips to better understand, evaluate, and eventually purchase the best technology for your needs. Contents What is binding affinity? ......................................................................................................1 Why measure binding affinity? ............................................................................................2 When do researchers measure binding affinity? ................................................................3 What tools are available to measure binding affinity? .......................................................4 Technologies for measuring binding affinity ......................................................................9 How do I evaluate my research needs? ...............................................................................9 How can I find the right solution? .....................................................................................10 How do I evaluate vendors? ...............................................................................................11 How do I make an informed decision? ..............................................................................13 How do I acquire the technology? .....................................................................................13 Total cost of investment ....................................................................................................15 Buyer’s checklist .................................................................................................................16 Resources for binding affinity ............................................................................................17 What is binding affinity? While it's very common for biologists and chemists to test whether or not two molecules interact with each other, it's much more useful to gather information on the nature of that interaction. How strong is it? How long will it last? What does this mean for its biological function? These questions can be answered by studying binding affinity. Binding affinity is the strength of the interaction between a single biomolecule and its binding partner, or ligand. It can be quantified, providing information on whether or not molecules are interacting as well as assigning a value to the affinity. Typically, when measuring binding affinity, you’re interested in several parameters, but mostly in the unit of measurement called the dissociation constant (Kd), which defines the likelihood that an interaction between two molecules will break. The smaller the dissociation constant, the more tightly bound the ligand is and the higher the affinity is between the two molecules. “The dissociation constant (Kd ) defines the likelihood that an interaction between two molecules will break and is a useful measurement to quantify binding affinity.” Binding affinity is an important metric used in both academia and industry. Academic researchers study binding affinity to learn about structural biology, structure-function relationships, and the intermolecular interactions that drive biological processes. On the other hand, drug developers study binding affinity to identify high-affinity molecules that bind to drug targets selectively and specifically. In this case, affinity can guide decisions about the biological relevance of a particular molecule, such as whether the molecule under investigation warrants further screening or characterization. 1 Why measure binding affinity? Almost every process in biology can be attributed to an interaction between molecules. With the thousands of individual molecules that make up a cell, researchers are challenged with determining which types of molecules interact with each other and figuring out the consequences of these interactions. Scientists use Kd to determine or “rank-order” binding reactions that may often translate into biological function or uncover the relevance of the targets being examined. The more researchers know about these interactions, the more they understand the biological systems in which they work with their intricate network of molecular pathways that control various cellular processes. Precisely characterizing biomolecular interactions in a biological system is an important cornerstone in basic research. In applied science, measuring the binding affinity of interactions is a prerequisite for the development of new products, such as drugs, enzymes or biomarkers. Measuring binding affinity has many applications, including identifying and screening small and/or large molecules, monitoring the regulation of cellular pathways, screening compound and drug candidates, testing structure-function relationships, and optimizing the development of assays that examine the interaction of two molecules. 2 “Precisely characterizing biomolecular interactions in a biological system is an important cornerstone in basic research.” When do researchers measure binding affinity? Now that you understand the what and why of binding affinity, it’s time to discuss when it comes into play in the re- search workflow. Primarily, when you are interested in finding out if two molecules interact in a pathway or process of interest, you use binding affinity assays to see how they interact or bind to each other. You can also measure binding affinity when modifying a molecule as a way to see how changing its binding properties relates to the pathway or process you are studying. Binding affinity is also useful when you need MEASURING BINDING AFFINITY IS USEFUL FOR: to develop a functional assay to monitor a pathway, • Characterizing receptor binding properties as you may need to measure binding as part of the assay. • Measuring interactions with antibodies • Analyzing protein complexes Academic researchers typically want to understand • Investigating enzyme inhibition the biology and regulation of a target molecule that • Observing molecular transport processes may or may not have any therapeutic potential. For • Mapping epitopes example, to understand a molecular pathway, it is • Optimizing leads important to be able to selectively modify molecules • Pursuing fragment-based lead discovery and quantitatively determine how these modifications • Measuring the effects of buffers, solutions, and concentration influence the overall pathway. on binding affinity 3 On the industry side of the research spectrum, binding affinity is a useful metric during the earliest parts of the drug development process when scientists are screening for any compounds that interact with their target of interest. Beginning with a large library of compounds or ligands, industry scientists begin the screening process by identifying which ones bind to the target protein, and then continue with an increasingly smaller pool of compound candidates. After they complete much of their pre-clinical work, researchers may measure binding affinity to determine and rank various compounds’ binding affinities for the target protein as an indicator of potency of a possible drug candidate. What tools are available to measure binding affinity? QUALITY COUNTS When it comes to measuring binding affinity, or performing Scientists use many different tools to measure binding any other experiment, for that matter, the quality of your affinity, although most of them fall into one of two results will depend on the quality of the source material. categories: qualitative methods and quantitative methods. If you don’t have any data on the quality of your source Qualitative methods such as ELISAs, pull-down assays, material, the experiment is less likely to succeed. and gel shift assays work by immobilizing one protein to a substrate and applying another protein (typically containing a label or reporter tag) to it. If the two proteins bind, they release a detectable signal. These methods merely provide a yes/no answer as to whether binding occurred. These techniques may be suitable for labs that occasionally analyze protein interactions or are examining protein interactions at a very gross level. In contrast, quantitative methods provide a scalar readout of binding affinity, releasing a signal that indicates the strength of the interaction. Biosensor-based methodologies work by immobilizing a binding partner to a surface and presenting the test partner to interact with it. The change in signal is observed and recorded by the instrument. Other quantitative methods use capillaries or tubes instead of immobilizing proteins to a surface. In this section, we’ll walk through the most common biophysical technologies and their strengths and weaknesses in terms of throughput, speed, sensitivity, and ease of use. 4 Surface Plasmon Resonance Surface plasmon resonance (SPR) is a common Strengths Additionally, SPR measures changes in biosensor-based technique for measuring SPR determines binding kinetics and refractive index. Any buffer component that biomolecular interactions. constants using label-free detection, which influences the refractive index of a sample eliminates the need for dyes and tags and (e.g. DMSO, a common solvent for small How it works allows for higher-sensitivity measurements. molecules) can cause artifacts in SPR SPR is a surface-based biosensor
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